1. Field of the Invention
The present invention relates to a novel aldolase that produces 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG), which is a precursor of monatin, and methods for producing 4R-IHOG and 4R-monatin by the use thereof.
2. Discussion of the Background
4-(Indole-3-ylmethyl)-4-hydroxy-2-glutamic acid(3-(1-amino-1,3-dicarboxy-3-hydroxy-butane-4-yl)indole (hereinafter referred to as monatin) represented by the following structural formula is found in the root of the plant, Schlerochiton ilicifolius, and because of its remarkably high sweetness intensity is a compound that is anticipated to be a sweetener with a low caloric value (see JP-P-64-25757-A).

As shown in the foregoing structure, monatin has two asymmetric carbon atoms at positions 2 and 4. The naturally occurring stereoisomer thereof was reported to be a (2S, 4S) isomer. Three additional stereoisomers are possible, and it has been confirmed that each of them has sweetness intensity that is several ten to several thousand times greater than that of sucrose (Table 1).
TABLE 1OpticalSweetnessIsomer(vs. Sucrose)2R, 4R2700 times2R, 4S1300 times2S, 4R 300 times2S, 4S 50 times
As is shown in Table 1, in addition to the naturally occurring (2S, 4S)-monatin the other stereoisomers have the sweetness intensity with high scale factor. Particularly, (2R, 4R)-monatin has a remarkably high sweetness intensity, which is 2,700 times greater than that of sucrose. As such, the (2R,4R) stereoisomer is particularly promising as a sweetening agent or a sweetening agent ingredient (sweetener). Therefore, a critical demand exists for the development of a method for efficiently producing monatin with high content of (2R, 4R)-monatin.
To address this demand, the present inventors have developed a new method for synthesizing monatin by performing the following reactions (a) and (b) using commercially available indole pyruvic acid and pyruvic acid as reagents (International Publication No. 03/056026 Pamphlet).
(a) Reaction step of synthesizing a precursor keto acid (IHOG) by aldol condensation of indole pyruvic acid and pyruvic acid (or oxaloacetic acid).
(b) Reaction step of aminating position 2 of IHOG.

International Publication No. 04/018672 Pamphlet discloses aldolases derived from Pseudomonas taetrolens and Pseudomonas coronafaciens as enzymes that are useful for producing the precursor keto acid (IHOG) from indole pyruvic acid and pyruvic acid (or oxaloacetic acid) in the aldol condensation of (a) in the aforementioned synthetic route of monatin. These aldolases have also been found to catalyze a reaction for the production of a keto acid such as 4-phenylmethyl-4-hydroxy-2-oxoglutaric acid (PHOG) in addition to IHOG.
There are two isomers, 4R-isomer and 4S-isomer in IHOG. In order to efficiently produce the (2R, 4R)-monatin (i.e., the isomer with highest sweetness), it is desirable to preferentially produce IHOG of a 4R-isomer (4R-IHOG) (4S-isomer is referred to as 4S-IHOG) in the aldol condensation reaction of (a) in the aforementioned synthetic route of monatin, and obtain 4R-isomer-rich IHOG. A chiral molecule often exhibits a physiological activity that is different by each isomer, and it is also likely that IHOG exhibits different characteristics by each isomer. Thus, separately produced 4R— and 4S-isomers may be utilized for uses other than the use as the monatin precursor. Therefore, it would be highly beneficial to industry to develop a method for producing preferentially one isomer of IHOG (e.g., one of 4R-IHOG and 4S-IHOG).